Biofertilizing Bacterial Strain

ABSTRACT

The present invention relates to the field of crop fertilization, and particularly to a biofertilizing bacterial strain. In particular, the present invention relates to the bacterial strain deposited on Oct. 24, 2018, at the Collection Nationale de Culture de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCM I-5373, and to the uses of this strain. The invention also relates to a composition comprising the above-mentioned bacterial strain and to a fertilization process comprising the application of this composition to a plant or to a soil.

TECHNICAL FIELD

The present invention relates to the field of crop fertilization, andparticularly to a biofertilizing bacterial strain.

PRIOR ART

The second half of the 20^(th) century saw a significant intensificationof agriculture in developed countries. This intensification was largelybased on the use of a set of inputs which allowed better control of thefactors limiting agricultural production (Tilman et al., 2002). Thewidespread use of chemical fertilizers provides much of the explanationfor the very rapid growth of agricultural production from the 1950sonwards. However, the intensive use of mineral nitrogen (N) fertilizershas led to the transfer of these elements to surface and groundwater andthe emission of greenhouse gases that contribute to the destruction ofthe ozone layer (Binbradan et al., 2015; Sebilo et al., 2013). Inaddition, N fertilizers have contributed to altering biologicaldiversity in agrosystems, with consequences for ecosystem functioning(Tilman et al., 1997) and primary productivity (Hector et al., 1999).These socially-unacceptable negative effects result mainly from poor Nfertilizer use efficiency by crops. For example, 20-80% of thesefertilizers are said to be dispersed in the environment rather thanbeing absorbed and assimilated by crops (Bindraban et al., 2015). Today,it is therefore necessary to limit the use of chemical N fertilizers.

For an element such as sulfur (S), the problem is different. Indeed, theimplementation since the 1980s of international directives to reduceindustrial atmospheric emissions and the reduction of the free S contentof N, P and K fertilizers has paradoxically led to the appearance of Sdeficiencies in crops. These deficiencies can be detrimental to cropyields and quality. It is therefore necessary to improve the supply of Sfor crops, especially since S improves the assimilation of N in plants.

There is therefore a genuine need for non-chemical solutions, based onbiological processes, that improve the availability of soil nutrients(mineral elements) to the plant (biofertilizer solution) and increasethe efficiency of their use by the plant (phytostimulant solution). Thisis particularly important for N and S insofar as more than 90% of N andS in soils are in organic forms that must be transformed by soilmicroorganisms (process of decomposition and mineralization of organicmatter) to release mineral forms accessible to plants.

SUMMARY OF THE INVENTION

The present invention relates to a biofertilizing bacterial strain formeeting the above needs.

Thus, in a first aspect, the present invention relates to a bacterialstrain as deposited on Oct. 24, 2018, at the Collection Nationale deCulture de Microorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX15, under the Budapest Treaty under number CNCM I-5373.

According to another aspect, the present invention relates to acomposition comprising the bacterial strain as deposited on Oct. 24,2018, at the Collection Nationale de Culture de Microorganismes (CNCM),28 rue du Dr. Roux, 75724 PARIS CEDEX 15, under the Budapest Treatyunder number CNCM I-5373.

The invention also relates to a fertilization process, said processcomprising a step in which the composition in accordance with theinvention is applied to a plant or to a growing medium such as soil.

In another embodiment, the present invention also relates to the use ofthe above-mentioned bacterial strain as biofertilizer or phytostimulant.

DISCLOSURE OF THE INVENTION

The present inventors have succeeded in identifying and isolating abiostimulant bacterial strain that exhibits remarkable biofertilizerproperties and solves the above problems.

“Biostimulants” are defined as products containing one or moresubstance(s) and/or microorganisms whose function consists, when appliedto plants or in the rhizosphere, in stimulating natural processes forthe benefit of nutrient uptake and/or increased efficiency of nutrientuse, as well as tolerance to abiotic stress factors, independently ofthe nutrient content of the products (Du Jardin, 2012). Depending on thebiological processes influenced by the biostimulant, it can be describedas a “phytostimulant,” “biofertilizer,” “soil life activator,” or“phytoactivator” (Faessel and Tostivint, 2016).

Biostimulants therefore include solutions based on microorganisms(microbial inoculants) or organic substances of various chemical natures(Calvo et al., 2014). Microbial inoculants include free microorganisms(bacteria and fungi) and/or symbiotic microorganisms (bacteria belongingto the Rhizobiaceae, mycorrhizal fungi). Some free bacteria that can beused as microbial inoculants, can be described as plant growth-promotingrhizobacteria (PGPR) (Hayat et al., 2010).

Microbial inoculants, including PGPR, can enhance plant growth byincreasing the availability of specific mineral elements in the soil,namely phosphorus (P), potassium (K), iron (Fe), calcium (Ca), andmagnesium (Mg) (Calvo et al., 2014). Microorganisms act by i)solubilizing elements such as P via the production of organic acids, ii)mineralizing organic P in the soil via phosphatase production, iii)producing siderophores.

A “biofertilizer” bacterial strain has the capacity to stimulate thebiological functioning of the soil in relation to the mineralization oforganic matter and to improve the availability of mineral elements, inparticular N, S and even P. This capacity can result from the productionof microbial enzymes for the decomposition and mineralization ofelements, such as for example an arylsulfatase activity which releasesmineral S or protease/aminopeptidase activities which release N.

A bacterial strain with the capacity to modify the microbial balance insoils and to improve the mineralization of organic matter globally isdescribed as a “soil life activator”.

A bacterial strain is also called “phytostimulant” when it has thecapacity to stimulate the growth, in particular the root system, ofplants in their early stages of development. This type of straintherefore improves soil exploration and element interception capacitiesby the plant. Phytostimulant strains can influence plant rootdevelopment via the production of phytohormone analogues that regulateroot initiation and elongation and the production of absorptive hairs(Vacheron et al., 2013).

The bacterial strain in accordance with the present invention has verystrong biofertilizer, phytostimulant and soil life activatorsactivities. Indeed, the present inventors have demonstrated inparticular that the inoculation of the bacterial strain in accordancewith the invention significantly increased the enzymatic activities ofthe soil involved in the decomposition and mineralization of N and S(and particularly the protease and arylsulfatase activities).

The inventors have also demonstrated that the strain according to thepresent invention significantly increases the root biomass, thuspotentiating a better capture of soil minerals by the plant.

The bacterial strain in accordance with the invention is the straindeposited on Oct. 24, 2018, at the Collection Nationale de Culture deMicroorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX 15, underthe Budapest Treaty under number CNCM-I-5373.

After analysis (partial sequence of the 16S rDNA with a length of 1374bp amplified with Taq PrimeStar Max), it turns out that the presentstrain is affiliated with the genus “Microbacterium” and hasparticularly 98% identity with Microbacterium resisters strain DMMZ 1710(GenBank accession no.: NR 026437.1). In the context of the presentapplication the bacterial strain constituting the subject matter of theinvention may therefore also be referred to as a “M” or“Microbacterium-affiliated” strain.

The bacterial strain in accordance with the present invention may beformulated as a composition. Thus, in another aspect, the presentinvention relates to a composition or “inoculant”, comprising thebacterial strain in accordance with the present invention.

The composition may be in powder, granule, cream (or “mud”) or liquidform (Bashan et al., Inoculants of plant growth-promoting bacteria foruse in agriculture; Biotechnol Adv 16:729-770, 1998; Catroux et al,Trends in rhizobial inoculant production and use, Plant Soil 230:21-30,2001). In a preferred embodiment, the bacterial strain in accordancewith the invention is formulated in liquid form.

The person skilled in the art knows several ways to formulatebiofertilizer bacterial compositions, or “inoculants” (see for exampleBashan, Yoav, et al. “Advances in plant growth-promoting bacterialinoculant technology: formulations and practical perspectives(1998-2013).” Plant and Soil 378.1-2 (2014): 1-33).

In order to prepare such a composition, the bacterial strain willtypically be incorporated into a carrier material. The carrier mustallow the growth of the bacteria in accordance with the invention,maintain a satisfactory level of viable bacteria for a given period oftime, and allow the release of these bacteria in sufficient amounts toallow their biostimulant effect (see Date et al., “Advances in inoculanttechnology: a brief review”, Aust J Exp Agr 41:321-325, 2001). Thiscarrier can be in solid, liquid or gel form. Carriers can be dividedinto five main categories:

-   -   soils including peat, charcoal, biochar, clays, and inorganic        soils;    -   organic plant and animal wastes of industrial or agricultural        origin;    -   inert materials such as polymers (like alginate or chitosan) or        rock fragments (like vermiculite and perlite);    -   freeze-dried or dehydrated microbial cultures incorporated into        a solid carrier or used as such; and    -   liquid carriers that comprise the bacterial strain to be        formulated alone or in the presence of certain additives to        improve stability, dispersion or turbidity.

The carrier may be pre-sterilized and enriched with nutrients such assucrose, maltose, trehalose, molasses, glucose and glycerol. Thisincreases the viability and shelf life of the composition comprising thebacteria in accordance with the invention.

“Soil” carriers may include additives. These additives are typicallyselected from chitin, pyrophyllite, coal, sedimentary rocks such aslignite, sugars (monosaccharides, oligosaccharides and polysaccharides),gum arabic and mineral and organic oils.

Additives used for liquid carriers are typically selected fromcarboxymethyl cellulose, glycerol, sugars (monosaccharides such asglucose, galactose, fructose; oligosaccharides and polysaccharides suchas sucrose, lactose, maltose or trehalose), glycerol, ferric EDTA, gumarabic and mineral and organic oils.

The composition may contain only the bacteria in accordance with theinvention, in which case it is referred to as a “primitive” inoculant.

Compositions in liquid form comprise on average at least 10⁶ CFU per mL.These inoculants are typically formulated as a bacterial suspension in asolution containing water and/or mineral oils and/or organic oils.

The composition can also be in solid form such as powder or granules.According to this formulation, the bacteria may be encapsulated inpolymeric matrices such as alginate beads/granules. Typically, thebacteria are formulated in a carrier consisting of inert materials (suchas alginate) and the mixture is made into a granular or powder form. Theformulation can then be freeze-dried. Typically, freeze-drying can beperformed in the presence of a cryoprotectant such as sucrose (forexample present in an amount between 0 and 10%, specifically 5% byweight).

The bacterial strain or composition in accordance with the presentinvention can be used in a soil fertilization process. Thus, in anotheraspect, the present invention relates to a fertilization process, saidprocess comprising a step in which the composition or the bacterialstrain in accordance with the invention is applied to a plant or togrowing medium such as soil.

A “fertilization process” aims at providing a growing medium with themineral elements necessary for the development of a plant. The growingmedium can be the soil, but also a growing support (for all marketgardening applications, whether soilless or not) such as potting soils,but also supports for aquaponics, hydroponics or aeroponics. Therefore,“fertilization”, as used herein, means both the fertilization of cropsand the amendment of uncultivated soil.

In the context of the present invention the soil can be any type of soilaccording to the international soil classification system (FAO-WorldReference Base).

The “plant” can be any living being belonging to the plant kingdom,whether cultivated or not. The plant is a terrestrial plant. It can be awoody plant or an herbaceous plant. The plant can, for example, beselected from the Poaceae, leguminous plants (Fabaceae) or oil-seedplants.

In a particular embodiment, the plant is a cereal selected from thegroup consisting of maize (Zea mays L.), rice (Oryza sativa, Oryzaglaberrina), the various species of wheat (Triticum aestivum; Triticumspelta; Triticum durum; Triticum dicoccum; Triticum turgidum L. subsp.turanicum; Triticum monococcum), barley (Hordeum vulgare L. subsp.vulgare; hordeum hexastichum); sorghum (Sorghum bicolor); oats (Avenasativa), millet (Pennisetum glaucum; Panicum milliaceum; Setariaitalica; Panicum sumatrense), rye (Secale cereale), triticale(Triticosecale).

The “plants” are typically crops:

-   -   field crop plants are typically selected from peanuts, spring        and winter oats, fodder and industrial beets, soft winter and        spring wheat, durum wheat, bromegrass, orchard grass, spelt,        fescue, timothy, grain, forage and silage maize, millet,        miscanthus, moha forage and grain, spring and winter barley,        panic grass, English and Italian ryegrass, rice, spring and        winter rye, sorghum, switchgrass, spring and winter triticale,        spring and winter camelina, hemp, winter and spring rapeseed,        canola, spring and winter white and brown mustard, turnip rape,        pastel, spring and winter fava bean, bird's-foot trefoil, lupin,        alfalfa, spring and winter peas, forage and protein peas,        sainfoin, soybeans, white, red and red clover, vetch, hops,        buckwheat, spring and winter flax (oilseed and textile), tobacco        and sunflower;    -   plants grown in fallow are typically selected from English and        Italian ryegrass, Persian clover, white mustard, phacelia,        white, red or purple clover and vetch;    -   vegetable crops are typically selected from garlic, dill,        artichoke, asparagus, eggplant, beet, broccoli, cardoon, carrot,        celery root and stalk, tuberous chervil, chicory, Chinese        cabbage, Brussels sprouts, leafy cabbage, head cabbage,        cauliflower, kohlrabi, watercress, cucumber, gherkins, zucchini,        melon, watermelon, pumpkin, shallot, spinach, fennel, fava bean,        flageolet, strawberry, beans, lettuce, dry and fresh lentils,        maize, turnip, cowpea, onion, parsley, chili, leek, peas, bell        pepper, potato, purslane, radish, horseradish, rhubarb, arugula,        rutabaga, salsify, curly endive, black salsify, tomato and        Jerusalem artichoke;    -   fruit crops such as lemon, clementine, lime, mandarin, orange,        grapefruit, quince, fig, almond, chestnut, hazelnut, walnut,        apricot, cherry, jujube, cherry-plum, nectarine, peach, plum,        kiwi, nashi, medlar, olive, cranberry, black currant, rose hip,        raspberry and other Rubus, currant, blackberry, blueberry, black        elder, pear and apple;    -   seed-bearing crops are typically selected from beet, brome,        cocksfoot, fescue, ryegrass, seed-bearing forage legumes,        seed-bearing umbellifers, seed fennel, seed bean, seed lupin,        seed trefoil, seed alfalfa, seed maize, hemp, flax,        Chrysanthemum, bitter apple, wallflower, carnation, pansy, sweet        pea, queen daisy, hollyhock, beet, carrot, celery, annual and        biennial chicory, cabbage, chives, cucurbits, shallot, spinach,        beans, lettuce, lamb's lettuce, turnips, onions, parsnips,        parsley, leeks, peas, chickpeas, radishes, arugula, dill,        chervil, coriander and clover;    -   plants cultivated in ornamental crops are typically selected        from conifers, broad-leaved trees, elms, lilies, Hippeastrum,        Narcissus, nerine, iris, hyacinth, lily, lily of the valley,        gladiola, Hydrangea, carnation, pale iris, palm, Pelargonium,        roses, tulips and other floral species;    -   plants cultivated in tropical crops are typically pineapple and        other anacardiaceous, avocado, banana, sugar cane, passion        fruit, Chinese cabbage, cassava, sweet potato, yam, mango and        Papaya;    -   plants grown for viticulture such as wine or table grapes        (including the various species of vines); and    -   plants cultivated for aromatic, perfume or medicinal purposes        are typically selected from wormwood, yarrow, mugwort, costmary,        burdock, dill, Roman chamomile, spices, Angelica, basil,        caraway, chervil, chives tarragon, bay leaves, rosemary, mint,        parsley, sage, gentian, lavender and lavandin, lovage, evening        primrose, oregano, borage, poppy seed, safflower, goosefoot,        squash, castor oil, sesame, savory, marigold and thyme.

The “plant” in accordance with the invention may be selected from any ofthe crop species listed above, and particularly from the differentvarieties of these species.

Preferably, the “plants” in accordance with the invention are fieldcrops, vegetable crops (including soilless) and viticulture, as definedabove.

The application can be performed at any stage of plant development. Theapplication can for example be performed directly on the seeds (forexample by coating just before sowing or with precoated seeds withmixtures of carboxymethyl cellulose and starch as a coating base), onthe soils to be cultivated or on the liquid or solid culture carriers(before or after sowing), for example by distribution of the inoculum inthe seed line (for example by mixing the inoculum with claymicrogranules distributed in the seed line or by direct inoculation onseeds using adhesive liquids), directly on the plant by foliar sprayingor directly on a plant explant or on a cutting.

Typically, when applied directly to the seed, the composition is mixedwith the seed by hand, in a rotary drum, or in a mixer. This mixing mayrequire the addition of adhesives, such as gum arabic, carboxymethylcellulose, sucrose solution or vegetable oils, to ensure that the seedsare covered with the necessary number of bacteria (see for example SenooK, Narumi I, 2006. Carrier materials. p. 41-49; BiofertilizerManual.Japan Atomic Industrial Forum (JAIF)). After this mixing, the seeds areusually dried. Alternatively, the composition can be sprayed onto theseeds. The sprayed seeds are then sown after drying.

Typically, when the composition is applied directly to the soil to becultivated, it is in granular form and distributed in the seed line.Alternatively, when in liquid form, the composition can be sprayed ontothe soil to be cultivated.

Alternatively, when the growing medium is a support for soillessculture, such as aeroponics, hydroponics, or aquaponics, the compositioncan be directly mixed with the culture substrate, for example in theform of peat.

Typically, the application will be done at sowing time (by applicationon the soil or by seed coating) or by inoculation of the culturesubstrates for soilless crops.

According to another embodiment, the present invention also relates tothe use of the bacterial strain deposited on Oct. 24, 2018, at theCollection Nationale de Culture de Microorganismes (CNCM), 28 rue du Dr.Roux, 75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCMI-5373 as biofertilizer.

The invention also relates to the use of the bacterial strain depositedon Oct. 24, 2018, at the Collection Nationale de Culture deMicroorganismes (CNCM), 28 rue du Dr. Roux, 75724 PARIS CEDEX 15, underthe Budapest Treaty under number CNCM I-5373 as phytostimulant.

The following examples illustrate the invention in greater detail butshould not be construed as limiting its scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the protease activity of the soil after inoculationwith the strain in accordance with the invention;

FIG. 2 represents the nitrate content in the soil after inoculation withthe strain in accordance with the invention;

EXAMPLES Example 1: Analysis of the Biofertilizer Properties of theStrain of Interest on Soil Microcosm (Controlled Conditions)

Materials and methods: A “model” soil was taken from an agriculturalplot at the ENSAIA experimental farm. The soil (silty clay; OM 4.5%;total N 0.3%; C/N 13.8; pH_(water) 6.5) was sieved to 5 mm and thenstored at room temperature until use. Soil microcosms were made byplacing 70 g of soil at 60% of its water holding capacity in 0.5 L LeParfait jars. The microcosms were pre-incubated for 2 weeks at 20° C.and then inoculated with 5 mL of 7*10⁹ CFU bacterial suspension (i.e.,10⁸ CFU/g fresh soil) of the selected bacterial strain. Controlmicrocosms were treated with 5 mL of sterile saline. The soilmicrocosms, at 80% water holding capacity, were then incubated at 20° C.in the dark. Soil samples were taken at 0, 3, 7, 14 and 28 days afterinoculation. Four replicates per treatment and sampling time wereperformed. At each sampling time, a set of variables were measuredwithin 48 h after sampling, namely (i) soil microbial enzymaticactivities in relation to N, S and P dynamics, (ii) measurements ofwater-soluble C and N contents, (iii) microbial C and N biomass, (iv)mineral N, S and P contents and (v) soil amino acid contents. Soilsamples were immediately frozen at −20° C. for DNA extractions and 16SrDNA gene abundance measurements.

Potential arylsulfatase activity in the soil was measured according tothe protocol of Tabatabai and Bremner (1970). Protease potentialactivity was measured according to the protocol of Ladd and Butler(1972). Potential phosphatase activity was measured according to theprotocol of Dick et al. (1996). Finally, potentialleucine-aminopeptidase activity was measured according to the protocolof Spungin and Blumberg (1989).

To estimate the soluble organic N and mineral N contents of the soils,10 g of fresh soil was extracted with 50 mL of 1 M KCL for 45 min usinga revolution mixer. Extracts were filtered through Whatman No. 42 filter(Harper, 1924) and stored at −20° C. until analysis. The mineral Ncontents (NO₃ ⁻ and NH₄ ⁺) of the extracts were determined using a SAN++CFA molecular absorption spectrophotometer (Skalar Analytical, Breda,The Netherlands) by the INRA, Nancy, analysis unit. Amino acids werequantified according to the protocol of Darrouzet-Nardi et al. (2013).Soluble C and N were extracted with hot water as described by Vong etal. (2007). The C and N content of the hot water extracts was determinedwith a TOC-V CHS (Shimadzu, Kyoto, Japan).

SO₄ ²⁻ were extracted from the soil by revolution mixing of 10 g of soilin 50 mL of 16 mM KH₂PO₄ for 45 min (Walker and Doornenbal, 1972) andthen filtered a first time on Whatman No. 42 paper and a second time ona 0.45 μm pore size filter. SO₄ ²⁻ from the KH₂PO₄ extracts weresubsequently determined by ion chromatography (Dionex).

PO₄ ²⁻ were extracted by revolution mixing of 5 g of soil in 50 mL of0.5 M NaHCO₃ for 45 min and then filtered through Whatman No. 42 paperfollowing the protocol of Hedley et al. (1982). PO₄ ²⁻ from the NaHCO₃extracts were subsequently determined according to the protocol ofIrving and McLaughlin (1990).

Microbial C and N biomasses were measured by the fumigation-extractionmethod (Vance et al., 1987). C and N concentrations of fumigated andunfumigated 0.5 M K₂SO₄ extracts filtered on Whatman No. 42 weredetermined with a TOC-V CHS (Shimadzu, Kyoto, Japan). Microbialbiomasses were determined by difference between fumigated andunfumigated extracts and division by an extraction coefficient of 0.45for microbial C biomass (Vance et al., 1987) and 0.54 for microbial Nbiomass (Brookes et al., 1985).

Results: Under controlled conditions, the Microbacterium-affiliatedstrain (M) stimulates certain soil microbial activities involved in themineralization of organic matter, without changing the size of the soilmicrobial biomass. Thus, microbial activities related to N decompositionare significantly higher in inoculated soils compared with uninoculatedsoils. Over 28 days of incubation, protease activity, which is involvedin the breakdown of protein, the main form of organic N in soils, was onaverage 50% higher in soils inoculated with the M strain compared withuninoculated soils (FIG. 1). Leucine aminopeptidase activity, whichdegrades peptides released by proteases into amino acids, was increasedby 3% to 23% in soils inoculated with the M strain from 14 days afterinoculation. Arylsulfatase activities that mineralize sulfate esters, amajor and among the most labile form of organic S in soils, are alsostimulated in inoculated soils, weakly 3 days after inoculation (+9%activity in soils inoculated with the M strain) and strongly 14 daysafter inoculation (+109% activity in soils inoculated with the Mstrain).

The M strain improves the availability of mineral N in the form of soilnitrate (FIG. 2), which is the main source of N for plant nutrition.

Example 2: Analysis of the Phytostimulant Properties of the Strains ofInterest (Controlled Conditions)

Materials and methods: A first experiment under gnotobiotic conditionswas set up to study the effect of the bacterial M strain on the growthand root architecture of maize seedlings (Pioneer Hi Bred). The seedsused were surface sterilized. To this end, the seeds were successivelyplaced in the presence of 70% ethanol for 5 min and 5% sodiumhypochlorite for 10 min. The seeds were then rinsed 5 times with steriledistilled water. 100 μL of the last rinse water was spread on NB agarmedium to verify the efficiency of the surface sterilization.

Roughly 25 sterilized seeds were then germinated in 13.5 cm diameterPetri dishes on sterile Whatman No. 3 filter paper moistened with 10 mLsterile distilled water. The Petri dishes were placed in the dark for 3days at 28° C. Four pre-germinated seeds were then selected and arrangedin an equatorial line in a 13.5 cm diameter Petri dish. Each seed wasinoculated with 100 μL of bacterial suspension (10⁹ bacteria/seed) ofthe selected strain. The bacterial inoculum was prepared from a cultureat 10⁹ CFU/mL. One mL of culture was centrifuged at 10 000 rpm for 10min and then the pellet was taken up in 100 μL of sterile saline.Control pre-germinated seeds received 100 μL of sterile saline. Intotal, 4 replicates were performed per treatment. The Petri dishes wereplaced in a climatic chamber with a photoperiod of 16 h, a temperatureof 23° C. day and 18° C. night for 6 days. After incubation, root partswere collected. A set of root traits were analyzed. To this end, theroot system was spread in a thin layer of water, in a Plexiglas tank(1.5*20*30 cm), using fine tweezers. The root system was then scanned(Expression 1640XL scanner, Epson) and the images obtained were analyzedwith WinRhizo© software (Régent Instruments Inc., Quebec, Canada). Thetotal length (cm), the length of fine (diameter <2 mm) and coarse(diameter >2 mm) roots, the total surface area (cm²), the surface areaof fine and coarse roots and the average diameter (mm) of the roots wereestimated. After analysis, the roots were gently dried on filter paperand weighed to estimate the fresh mass. They were then placed in an ovenat 80° C. for 48 h to determine the dry mass.

A second experiment aimed at evaluating the effect of the M strain onthe growth, physiology and nutritional status of maize during the earlystages of development, as well as on the biological functioning and thecontent of mineral elements in the maize rhizosphere. The soil (siltyclay; OM 1.6%; total N 0.12%; C/N 7.8; pH_(water) 7.5) was collectedfrom an agricultural plot (Saint Martin des Champs, 77), air-dried andsieved to 5 mm. Before use, the soil was mixed with 10% (m/m) aquariumsand to facilitate subsequent root collection.

The inoculation of maize with the bacterial strain of interest wasperformed at sowing time, on sterilized and germinated seeds with 1 mLof an inoculum at a concentration of 10⁸ CFU/mL. Sterilized, germinatedbut uninoculated seeds were used as a control. One seed was placed at adepth of about 2 cm in a PVC tube (5*20 cm) containing 330 g ofsoil-sand mixture. This substrate was maintained at 80% of its waterholding capacity by weighing the tubes 5 times a week and watering, ifnecessary. At the 1-2 leaf, 3-4 leaf, 5 leaf and 5-6 leaf stages, 4tubes of each treatment modality were randomly selected and opened witha circular saw. After opening the tubes, the aerial parts were separatedfrom the soil-root system and collected. The roots were separated fromthe soil, collected with tweezers and then washed with tap water. Theaerial and root parts of the plants and the soil were stored at 4° C.until all variables were analyzed. An aliquot of soil was frozen at −20°C. for subsequent molecular analyses.

At the plant level, fresh mass and dry mass of aerial and root partswere measured. Root architecture was also analyzed as previouslydescribed (experiment under gnotobiotic conditions). At the level ofmaize rhizosphere soil, the variables of microbial abundance, microbialenzymatic activities and mineral element content were measured asdescribed in the context of the soil microcosm experiment.

Results:

In the second experiment, we analyzed the effect of the M strain on thegrowth and nutritional status of maize grown under controlled conditionsup to the 6-leaf stage as well as the biological functioning andavailability of mineral elements in the rhizosphere of this maize.

At the plant level, while the strain does not influence the biomass ofthe aerial parts of the maize or their height, it does alter rootgrowth. Thus, the fresh root biomass of plants inoculated with the Mstrain is 20% higher (p=0.01) than that of control plants at the 3-4leaf stage. The surface area of fine roots (diameter <2 mm) was greaterfor inoculated plants compared with the control. These results tend toconfirm the results obtained under gnotobiotic conditions.

Concerning the effects of M strain inoculation on rhizosphere soilfunctioning, measurements of microbial abundance, microbial enzymaticactivities involved in N, S and P dynamics and mineral element contentswere performed. The main effects of M strain inoculation on soil enzymeactivities related to N, S and P cycles were observed at the maize 3-4leaf stage. Arylsulfatase activity involved in S mineralization wassignificantly increased in the rhizosphere of plants inoculated with theM strain (+89%) compared with the control at the 3-4 leaf stage. At the5-6 leaf stage, this activity remained higher in inoculated soils (+18%for the M strain).

Example 3: Use of Molecular Markers to Discriminate the M Strain

The possibility of discriminating bacterial strains with RAPD-typemarkers was validated by comparing the profile of the M strain withother strains, in particular from the genus Pseudomonas (which is highlyabundant in natural environments).

The strains tested are listed in the table below:

TABLE 1 List of strains tested Strain code Strain name AchromoAchromobacter sp. Ps.chloro Pseudomonas chlororaphis Ps.sp Pseudomonassp. Ps.putida Pseudomonas putida Enterob Enterobacter ludwigii Ps-1Pseudomonas moraviensis Ps-2 Pseudomonas moraviensis Micro-1Microbacterium resistens (M strain) Micro-2 Microbacterium resistens (Mstrain)

Two RAPD-type markers were tested: M13 5′-GAGGGTGGCGGTTCT-3′ (SEQ IDNO: 1) and RAPD2 5′-AGCAGCGTGG-3′ (SEQ ID NO:2). Amplification reactionswere performed in a final volume of 12.5 μL in the presence of 15 ng DNAand 25 μM primers. Amplicons were separated on a 2% agarose gel toreveal the profiles.

Both markers are discriminating even against bacteria of the same genus.A better repeatability is observed for the M13 marker.

Example 4: Production of the M Strain

The scale-up was performed in an Applikon® bioreactor in a final volumeof 4 L. The medium used was composed of 10 g/L whole milk powder and 5g/L yeast extract. The tests will make it possible to determine the kLa(oxygen transfer coefficient), a parameter for scaling up (50 and then500 L bioreactors). The optimal temperature is 30° C. (see table below).

TABLE 2 Conditions for producing the strain Parameter Aeration StirringpH Temperature M. resistens M strain 2 vvm 150 rpm 6.9-7.1 30° C.

Tests conducted in 5 L fermenters produced microbial biomass of the Mstrain at an average of 1.11^(E+09) CFU/mL.

Example 5: Drying of the M Strain

The M strain shows very good survival to the freeze-drying process forthe conditions with cryoprotectant, even more marked for the conditionwith sucrose. The freeze-drying schedules are presented in Table 3. Inaddition, freezing the samples for freeze-drying had no impact on theviability of the bacteria. The aw of the samples is at most about 0.150and ensures good microbiological stability (viability and shelf life).The total duration of freeze-drying is 47 hours.

TABLE 3 Freeze-drying schedules Final temperature of the shelvesPressure Step duration Step (° C.) Ramp (min) (μbar) (min) Freezing −45Before loading Atmo 120 Primary −30 60 50 990 Drying −10 150 50 1350 10120 50 300 Secondary 20 90 50 60 Drying

With cryoprotectant, the loss of viability is negligible. Thus, apositive effect of cryoprotectant (5% sucrose) is observed. Withoutcryoprotectant, the survival of the strain remains very acceptable atabout half a log (Table 4).

TABLE 4 Strain viability Viability Viability Viability (CFU/mL)Viability after after Viability Loss of before before freeze- freeze-after freeze- viability freeze- freeze-drying drying drying drying (logCondition drying (logCFU/mL) (CFU/mL) (CFU/gDM) (logCFU/mL) reduction)The M strain 3.80E+10 10.58 1.03E+10 3.32E+11 10.01 0.57 withoutcryoprotectant The M strain 3.40E+10 10.53 2.48E+10 8.35E+11 10.39 0.14with 5% sucrose

1-4. (canceled)
 5. A fertilization process comprising a step in whichthe bacterial strain deposited on Oct. 24, 2018, at the CollectionNationale de Culture de Microorganismes (CNCM), 28 rue du Dr. Roux,75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCMI-5373, or a composition comprising said bacterial strain, is applied toa plant or to a soil.
 6. The fertilization process according to claim 5,wherein said plant is selected from field crop plants, vegetable cropplants and plants grown in viticulture.
 7. The fertilization processaccording to claim 5, wherein the application is made at sowing time, byapplication to the soil or by coating the seeds.
 8. The fertilizationprocess according to claim 5, wherein the application is made byinoculation of growing substrates intended for soilless cultivation.9-10. (canceled)
 11. The fertilization process according to claim 5,wherein said composition is in powder, granule, cream or liquid form.12. The fertilization process according to claim 5, wherein saidcomposition is in liquid form.
 13. The fertilization process accordingto claim 5, wherein said composition comprises the bacterial strain as asuspension in a solution containing water and/or mineral oils and/ororganic oils.
 14. A method for biofertilizing a soil, comprising a stepof applying the bacterial strain deposited on Oct. 24, 2018, at theCollection Nationale de Culture de Microorganismes (CNCM), 28 rue du Dr.Roux, 75724 PARIS CEDEX 15, under the Budapest Treaty under number CNCMI-5373 to a soil.
 15. A method for stimulating a plant growth,comprising a step of applying the bacterial strain deposited on Oct. 24,2018, at the Collection Nationale de Culture de Microorganismes (CNCM),28 rue du Dr. Roux, 75724 PARIS CEDEX 15, under the Budapest Treatyunder number CNCM I-5373 to a plant.